PROJECT SUMMARY/ABSTRACT Microglia, the brain?s resident immune cells, are essential for normal brain development and function, and likely involved in mental illnesses such as depression and schizophrenia. Because it is nearly impossible to obtain suitable human brain specimens, we are severely restricted from studying microglial function in these diseases, especially in vivo. This Mentored Career Development Award, by a clinician-scientist in the Departments of Psychiatry and Neurobiology at Stanford University, mentored by Dr. Ben Barres, will make the study of human microglia in mental illness a reality through related basic and translational research goals. The basic research goal is to advance our understanding of microglial biology by comparing the transcriptomes of microglia and blood-derived ?Microglia Like Cells? (MLCs) that enter the brain during disease. The translational goal is to use these results to generate human microglia from blood cells, allowing study of microglia without human brain tissue. This proposal outlines a 5 year mentored career development plan that harnesses the abundant educational and scientific resources at Stanford to accomplish the research aims described below while providing the candidate with needed training to function as an independent investigator. Preliminary data show that when mice lacking microglia due to knock-out of the CSF1 receptor (CSF1R) are transplanted with wild type (WT) bone marrow (BM), donor-derived cells fill the brain in a near-identical pattern to WT microglia, and express nearly all specific markers of microglial fate. These engrafted MLCs can be purified based on expression of Tmem119, a highly specific mouse and human microglial marker. RNAseq data shows that MLCs are highly similar to WT microglia. The proposed research will expand on these intriguing findings to better understand differences between MLCs and microglia, and create a reliable system for studying bone marrow-derived MLCs in vivo. Aim 1 will determine how closely MLCs resemble WT microglia, and in what ways they are different. Aim 2 will identify the specific hematopoietic cell types capable of generating MLCs. Aim 3 will translate these findings for use with human blood cells, by generating RNAseq profiles for highly pure resting human microglia, and comparing them to MLCs generated by transplantation of human BM into immune tolerant CSF1R knockout mice. This work will advance our understanding of core differences between microglia and MLCs, identify the transcriptomic signature of pure human microglia, and create a tractable system to study microglia using patient-derived hematopoietic cells, while fostering an independent research career centered around the role of microglia in mental health.